JOURNAL OF APPLIED TOXICOLOGY, VOL. 12(6), 439442 (1992)

Silymarin Protects against Paracetamol-induced Lipid Peroxidation and Liver Damage Pablo Muriel,? Tania Garciapina, Victor Perez-Alvarez a n d Marisabel Mourelle Departamento de Farmacologia y Toxicologia. Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Apartado Postal 14-740. Mexico D. F., CP 07000, Mexico

Key words: acetaminophen; lipoperoxidation; silymarin; liver damage.

The effect of silymarin on liver damage induced by acetaminophen (APAP) intoxication was studied. Wistar male rats pretreated (72 h) with 3-methylcholantrene (3-MC) (20 mg kg-' body wt. i.p.) were divided into three groups: animals in group 1 were treated with acetaminophen (APAP) (500 mg kg-' body wt. P.o.), group 2 consisted of animals that received APAP plus silymarin (200 mg kg-' body wt. p.0.) 24 h before APAP, and rats in group 3 (control) received the equivalent amount of the vehicles. Animals were sacrificed at different times after APAP administration. Reduced glutathione (GSH), lipid peroxidation and glycogen were measured in liver and alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGTP) and glutamic pyruvic transaminase (GPT) activities were measured in serum. After APAP intoxication, GSH and glycogen decreased very fast (1 h) and remained low for 6 h. Lipid peroxidation increased three times over the control 4 and 6 h after APAP treatment. Enzyme activities increased 18 h after intoxication. In the group receiving APAP plus silymarin, levels of lipid peroxidation and serum enzyme activities remained within the control values at any time studied. The fall in GSH was not prevented by silymarin, but glycogen was restored at 18 h. It was concluded that silymarin can protect against APAP intoxication through its antioxidant properties, possibly acting as a free-radical scavenger.

INTRODUCTION

Acetaminophen (paracetamol, APAP) is a relatively safe antipyretidanalgesic drug when administered at therapeutic doses but can produce hepatic centrilobular necrosis at toxic doses.' However, the events associated with its toxicity in the liver are largely unresolved. Theories such as binding of the reactive metabolite of APAP-N-acetyl-p-benzoquinone imine (NAPQ1)-to cellular macromolecules,2~3lipid pero~idation~,'and oxidation of critical sulphydryl groups and alteration of calcium homeostasish have been proposed. It has been demonstrated that paracetamol oxidation in the hepatocyte initiates a sequence of events that eventually leads to cell death.7 Antioxidants can inhibit these event^,^.^ suggesting that deleterious oxidative changes are involved. On the other hand, it has been reported that silymarin, a 3-oxyflavone occurring in the thistle Silybum marianum (L), Gaertn., protects the liver of experimental animals against several hepatotoxic substances, such as phalloidin, amanitin, galactosamine, ethanol and CC14.1h15 It appeared that hepatotoxic substances such as CCI4 and APAP become effective following their activation into a free-radical form. 16,' Moreover, CCl,-induced liver damage is lowered by substances that scavenge free r a d i ~ a l s . ' ~ . ' ~ J ~ On the basis of these considerations, we decided to prove the effect of silymarin on APAP-induced liver damage since it appears possible that silymarin might t Author to whom correspondence should be addressed. 026C-437x/92/06043%04$07.00

0 1992 by John Wiley & Sons, Ltd.

act by preventing, or inhibiting, lipid peroxide formation that can be induced by APAP through a radical m e c h a n i ~ m . ~Indeed, ~' the results reported in the present paper show that silymarin stiongly inhibits liver peroxide formation and damage induced by APAP intoxication. EXPERIMENTAL

Wistar male rats weighing 120-140 g and fed a purine chow diet ad libitum were divided into three groups. Animals in group 1 received a single i.p. dose of 3methylcholantrene (3-MC) (20 mg kg-' body wt.) dissolved in mineral oil; 72 h after 3-MC pretreatment, animals received an oral dose of APAP (500 mg kg-' body wt .) suspended in 1 YO carboxymethylcellulose (CMC) and delivered into the stomach through an intragastric tube. The second group received, in addition to 3-MC and APAP, an oral dose of silymarin (200 mg kg-' body wt.) suspended in 1% CMC 24 h before APAP intoxication. Animals in group 3 were used as the control group and received only the equivalent amount of the vehicles. Ten animals of each group were sacrificed at different times after APAP administration, as specified in the Results section. Animals were lightly anaesthetized with ether, blood was collected by heart puncture and the liver was rapidly removed. Serum was obtained for the following determinations: the activities of alkaline phosphatase (AP)," gamma-glutamyl transpeptidase (GGTP)19 and glutamic pyruvic transaminase (GPT).20 Liver pieces were separated for glycogen2* and malondialdehyde (MDA)22 determinations. Received 8 June 1991 Accepted (revised) 10 April 1992

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P. MURIEL E T A L I amp

I

t

Time ( h )

Figure 1. Effect of silymarin on the hepatic GSH depletion after 1, 3, 4, 6, 18 and 24 h of acetaminophen (APAP) intoxication. Each value represents the mean t SEM of ten animals in duplicate assays. +, Means different from the control group ( P < 0.05). *, Means different from the acetaminophen-treated group ( P < 0.05).

CQYTROL

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APAP t SlLYYARlN

+

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24

T i m e (h)

Figure 2. Effect of silymarin on the hepatic lipoperoxidation induced after 1, 3, 4, 6, 18 and 24 h of acetaminophen (APAP) intoxication. Each value represents the mean 5 SEM of ten animals in duplicate assays. +, Means different from the control group ( P < 0.05). *, Means different from the acetaminophentreated group ( P < 0.05).

For statistical analysis, TukeyZ3tests were performed to compare the values at different times against its control. Student's t-test was used in order to compare the group of APAP vs. the group of APAP + silymarin each time. In all cases a difference was considered to be significant when P < 0.05.

APAP APAP + S I L Y M A R I N

1

RESULTS A single dose of 500 mg kg-' body wt. of APAP was enough to produce diffuse areas of necrosis (ca. 60% of the liver tissue), as revealed by standard histological procedures, while livers from APAP + silymarintreated rats showed a similar structure to the controls (not shown). Reduced glutathione is one of the most important antioxidant molecules of the liver, and it is known that a decrease in ca. 70% of this compound does not allow the liver to maintain the normal redox state of the c e k 5 Figure 1 shows that APAP administration to rats pretreated with 3-MC induced a drastic decrease (ca. 70%) of liver GSH during the first 6 h of intoxication ( P < 0.05); 18 h after APAP administration, GSH levels recovered to above the control values ( P < 0.05). Silymarin pretreatment failed to preserve liver GSH. Blood GSH remained constant in all groups (data not shown). Malondialdehyde is a product of lipid peroxidation, therefore an increase in liver MDA levels indicates an increase in the degree of lipid peroxidation, a very well-known mechanism of liver damage.24 As shown in Fig. 2, APAP increased liver lipoperoxidation three times over control values ( P < 0.05) 4 and 6 h after APAP treatment; however, at 18 h MDA levels were normal. In the animals of the group treated with APAP + silymarin no significant changes were observed in MDA levels when compared to controls. Glycogen is the main source of liver energy; without it, the liver is not able to carry out energy-coupled hepatic functions. Figure 3 shows that the liver glycogen content decreased early (in the first hour) after APAP intoxication, reaching its lowest value (10% of control) after 4 h and remaining low for 24 h ( P < 0.05). In the animals of the group treated with APAP + silyma-

-

"

CONTROL

I

3

4

6

18

24

Time (h)

Figure 3. Effect of silymarin on the hepatic glycogen depletion after 1,3,4,6,18 and 24 h of acetaminophen (APAP) intoxication. Each value represents the mean 2 SEM of ten animals in duplicate assays. +, Means different from the control group ( P < 0.05). *, Means different from the acetaminophen-treated group ( P < 0.05).

rin the liver glycogen content also diminished within the first 6 h; however, after 18 h the liver glycogen content was completely restored ( P < 0.05) (Fig. 3). Alkaline phosphatase is an ectoenzyme of the hepatocyte plasma membrane; an increase in serum A P activity has been related to damage to the liver cell membrane.25 Gamma-glutamyl transpeptidase is an enzyme embedded in the hepatocyte plasma membrane, mainly in the canalicular domain; again, the liberation of this enzyme to sera indicates damage to the cell and thus injury to the liver. It is important to point out that serum GGTP activity is considered to be one of the best indicators of liver damage.26,27 Glutamic pyruvic transaminase is a cytosolic enzyme of the hepatocyte and an increase in the serum of this enzyme reflects an increase in plasma membrane permeability, which, in turn, is associated with cell death.26 The activities of AP (Fig. 4), GGTP (Fig. 5 ) and GPT (Fig. 6) were measured in sera. The three enzyme activities remained within the control values for 6 h after APAP intoxication; however, 18 h after APAP administration the three enzyme activities increased above the controls and remained high for 24 h ( P < 0.05). It is important to remark that in all

SILYMARIN EFFECT ON ACETAMINOPHEN TOXICITY

1

1

-

=-

i

APAP APAP

+ SILYMARIN

la

0

DISCUSSION

14 b

2;

2*

23

A

T i m e (h)

Figure 4. Time course of serum alkaline phosphatase activity after acetaminophen (APAP) intoxication. Each value represents the mean t- SEM of ten animals i n duplicate assays. f , Means different from the control group ( P < 0.05). *, Means different from the acetaminophen-treated group ( P < 0.05).

Time (hl

Figure 5. Time course of serum gamma-glutamyl transpeptidase activity after acetaminophen (APAP) intoxication. Each value represents the mean f SEM of ten animals in duplicate assays. +, Means different from the control group ( P < 0.05). *, Means different from the acetaminophen-treated group ( P < 0.05).

I

44 1

t --PAP

Figure 6. Time course of serum glutamic pyruvic transaminase activity. Each value represents the mean 2 SEM of ten animals in duplicate assays. f , Means different from the control group ( P < 0.05). *, Means different from the acetaminophen-treated group ( P < 0.05).

cases silymarin pretreatment completely preserved enzyme activities, indicating total protection.

Damage could be defined as the loss of the normal structure and function of the liver. Histological studies (not shown) revealed diffuse areas of necrosis in livers from APAP-treated rats, while livers from APAP + silymarin-treated rats showed a similar structure to the controls. Biochemical analysis of the APAP-intoxicated group showed significant increases ( P < 0.05) in serum markers of liver damage (AP, GGTP and GPT) and decreases in GSH and glycogen contents. The group receiving APAP + silymarin showed normal levels of most of the markers of liver damage. This indicates that histological analysis correlated well with the biochemical findings. In our model of APAP-induced liver injury, markers of damage in sera (AP, GGTP and GPT enzyme activities) reached a maximum 18 h after APAP administration. It is interesting to note that liver determinations (GSH, MDA and glycogen) were affected earlier than serum enzyme activity (AP, GGTP and GPT); this was probably due to the time required for the enzymes to be liberated into serum. In addition, the increase in serum enzyme activities could be the result of lipoperoxidative stress that occurred earlier (4 and 6 h after APAP administration). Reduced glutathione decreased very rapidly after APAP intoxication, probably because of NAPQI conjugation with GSH.'-3 On the other hand, liver glycogen decreased before lipid peroxidation increased, indicating that probably the fall in glycogen was not due to a lipoperoxidative process. One possibility is that the regulatory mechanisms of glycogen were uncoupled by covalent binding of NAPQI to a regulatory enzyme of glycogen; however, this cannot be inferred from the present data and more investigation is needed in order to clarify this point. The increase above control limits in liver GSH content 18 h after APAP administration was interpreted speculatively as a rebound owing to the low values of GSH at earlier times, i.e. 4 h. The liver can rapidly synthesize great quantities of GSH to prevent cell damage, as the amount of metabolites formed by the GSH conjugates can exceed the amount of GSH initially present in the liver by several-fold.2x It appears that, in the case of an overdose of acetaminophen, the phenomena of covalent binding and lipid peroxidation may well occur simultaneously in the liver cell following depletion of GSH. Membrane deterioration (and leak of cytosolic and membrane enzymes) due to extensive stimulation of lipid peroxidation could be an important facet of APAP t ~ x i c i t y , ~ and the observed inhibition of lipid peroxidation by silymarin may account in part for its beneficial activity. This protective action of silymarin is probably associated with its antioxidant properties, possibly acting as a free-radical scavenger even at low levels of GSH. In fact it has been demonstrated that silymarin is a good scavenger of superoxide and alkoxy radicals, as tested by the chemiluminescence technique.29 On the other hand, the calcium homeostasis theory6 is not in disagreement with the present data, since high levels of lipid peroxidation produce a change in

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the plasma membrane, which thus allows calcium accumulation.30 It is believed that GSH is one of the most important protective factors against oxidative damage. It may act not only as a scavenger by itself but also through some GSH-dependent enzymes. Fairhurst and co-workers5 have pointed out that the threshold for the onset of lipid peroxidation to occur appears to be at a GSH concentration of ca. 25% of the peak physiological concentration. We observed similar results in the present work, with an exception in the group receiving APAP + silymarin in which lipid peroxidation was prevented at low values of GSH, indicating that the beneficial effect of silymarin is not exerted by preserv-

ing liver GSH content but by preventing lipid peroxidation itself. This paper gives evidence that lipoperoxidative stress occurs during APAP-induced liver injury and that silymarin is capable of preventing both lipid peroxidation and liver damage.

Acknowledgements The authors want to express their gratitude to Ms Concepcih Avalos for secretarial assistance and Mr Alfredo Padilla for preparing the figures. We also thank Alejandro Nava, MD, for his critical review of the manuscript.

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